Thieno[2,3-d]pyrimidines with combined LH and FSH agonistic activity

ABSTRACT

The invention relates to thieno[2,3-d]pyrimidine derivatives according to general formula I, or a pharmaceutically acceptable salt thereof, 
                         
wherein N(R1)R2 are joined in a (2-6C)heterocycloalkyl ring. The compounds of the invention have LH as well as FSH receptor activating activity and can be used in fertility regulating therapies.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the national phase application under 35 U.S.C. § 371of PCT International Application No. PCT/EP02/09647, which has anInternational filing date of Aug. 29, 2002, and which designatedEuropean Patent Office Application Serial No. 01203327.0, filed Sep. 4,2001, as priority.

The invention relates to compounds having glycoprotein hormone agonisticactivity, in particular to compounds having both Luteinizing Hormone(LH) and Follicle Stimulating Hormone (FSH) agonistic activity. Theinvention furthermore relates to pharmaceutical compositions containingthe same as well as to the use of these compounds in medical therapy,particularly for use as a control of fertility.

Gonadotropins serve important functions in a variety of bodily functionsincluding metabolism, temperature regulation and the reproductiveprocess. The hypophyseal gonadotropins FSH and LH for example play apivotal role in the stimulation of follicle development and maturationwhereas LH is involved in induction of the ovulatory process (Sharp, R.M. Clin. Endocrinol 33, 787-807, 1990; Dorrington and Armstrong, RecentProg. Horm. Res 35, 301-342, 1979; Levy et al, Human Reproduction 15,2258-2265, 2000).

Currently, LH is applied clinically, in combination with FSH, forovarian stimulation i.e. ovarian hyperstimulation for in vitrofertilisation (IVF) and induction of ovulation in infertile anovulatorywomen (Insler, V., Int. J. Fertility 33 85-97, 1988, Navot andRosenwaks, J. Vitro Fert. Embryo Transfer 5, 3-13, 1988), as well as formale hypogonadism and male infertility.

Gonadotropins act on specific gonadal cell types to initiate ovarian andtesticular differentiation and steroidogenesis. The actions of thesepituitary and placental hormones are mediated by specific plasmamembrane receptors that are members of the large family of G-proteincoupled receptors. They consist of a single polypeptide with seventransmembrane domains and are able to interact with the Gs protein,leading to the activation of adenyl cyclase.

Gonadotropins destined for therapeutic purposes can be isolated fromhuman urine sources and are of low purity (Morse et al, Amer. J.Reproduct. Immunol. and Microbiology 17, 143, 1988). Alternatively, theycan be prepared as recombinant gonadotropins. In addition to theseproteins, gonadotropin receptors can be activated or deactivated bysynthetic low molecular weight compounds. Bicyclic heteroaromaticcompounds have been described in WO 00/61586. By in vitro and in vivoexperiments they are shown to be useful as LH agonists.

In normal females the release of pituitary LH and FSH is characterizedby a mid-cycle surge which precedes the ovulation. Ovulation ischaracterized by three distinct physiological phenomena i.e. oocytematuration, follicular rupture and luteinization. While the role of theLH-surge in the in vivo induction of these phenomena is undisputed, therole of the FSH-surge is less clear. However, it has been shown recentlythat FSH induces oocyte maturation in vitro by inducing cumulus cells toproduce a factor that positively overcomes hypoxanthine induced meioticarrest (Lu et al, Mol. Cell. Endocrinol. 164, 191-196, 2000). Thisfactor is thought to be a meiosis activating sterol (MAS).

In ovulation induction, it is desirable to provide the effects of LH asthe major component. According to the present invention compounds havebeen found with particular advantageous properties when used inprotocols for enhanced fertility. In these compounds LH activity isaccompanied by a FSH activity.

Thus the present invention provides low molecular weight compounds thatin addition to LH activity unexpectedly also have FSH activity. Ingeneral these compounds are thieno[2,3-d]pyrimidines which at the4-position of the pyrimidine ring are substituted by a phenyl groupwhich in turn is substituted at the meta position.

The present invention resides in thieno[2,3-d]pyrimidine derivativesaccording to general formula I,

or a pharmaceutically acceptable salt thereof, wherein N(R1)R2 arejoined in a (2-6C)heterocycloalkyl ring.

The most preferred compounds are tert-butyl5-amino-2-methylthio-4-(3-(2-(azetidin-1-yl)-acetamido)-phenyl)-thieno[2,3-d]pyrimidine-6-carboxamide;tert-butyl5-amino-2-methylthio-4-(3-(2-(morpholin-4-yl)-acetamido)-phenyl)-thieno[2,3-d]pyrimidine-6-carboxamide;tert-butyl5-amino-2-methylthio-4-(3-(2-(thiomorpholin-4-yl)-acetamido)-phenyl)-thieno[2,3-d]pyrimidine-6-carboxamide;tert-butyl5-amino-2-methylthio-4-(3-(2-(piperidin-1-yl)-acetamido)-phenyl)-thieno[2,3-d]pyrimidine-6-carboxamide;tert-butyl5-amino-2-methylthio-4-(3-(2-(pyrrolidin-1-yl)-acetamido)-phenyl)-thieno[2,3-d]pyrimidine-6-carboxamideand tert-butyl5-amino-2-methylthio-4-(3-(2-(piperazin-1-yl)-acetamido)-phenyl)-thieno[2,3-d]pyrimidine-6-carboxamide.

The term joined in a (2-6C)heterocycloalkyl ring in the definition ofFormula I, means that R1 and R2 together with the nitrogen atom to whichthey are bonded form a ring having 2-6 carbon atoms, optionallycontaining one or more heteroatoms selected from N, O and/or S. Examplesof such rings are azetidine, pyrrolidine, piperidine, piperazine,morpholine and thiomorpholine.

It has been shown that compounds of the above mentioned formula I showagonistic LH and FSH activity. In an in vitro bioassay using CHO cellsstably transfected with the human LH or FSH receptor, respectively, theEC₅₀ with regard to the LH receptor was found to be less than 5.10⁻⁸ Mwhereas with regard to the FSH receptor the EC₅₀ was less than 10⁻⁵M.Typically the FSH activity ranges from an activity of about 1% of the LHagonist stimulation to about 10% of the LH agonist stimulation.

The invention further resides in a pharmaceutical composition comprisinga thieno[2,3-d]pyrimidine derivative compound or salts thereof havingthe general formula I.

Thus, the compounds according to the invention can be used in therapy. Afurther aspect of the invention resides in the use of athieno[2,3-d]pyrimidine compound having the general formula I for themanufacture of a medicament for the control of fertility, morepreferably induction of ovulation. The present compounds are used toactivate both the LH and FSH receptors. The compound of the presentinvention can be used therefore in a method to treat females withfertility problems.

For therapeutic use, salts of the compounds of formula I are thosewherein the counterion is pharmaceutically acceptable. However, acidaddition salts of bases according to formula I, may also find use, forexample, in the preparation or purification of a pharmaceuticallyacceptable compound. All salts, whether pharmaceutically acceptable ornot, are included within the ambit of the present invention.

Examples of acid addition salts include those derived from mineral acidssuch as hydrochloric acid, phosphoric acid, sulphuric acid, preferablyhydrochloric acid, and organic acids like citric acid, tartaric acid,acetic acid, lactic acid, maleic acid, malonic acid, fumaric acid,glycolic acid, succinic acid, and the like.

Suitable administration routes for the compounds of formula I orpharmaceutically acceptable salts thereof, also referred to herein asthe active ingredient are intramuscular injections, subcutaneousinjections, intravenous injections or intraperitoneal injections, oraland intranasal administration. Preferably, the compounds may beadministered orally. The exact dose and regimen of administration of theactive ingredient, or a pharmaceutical composition thereof, willnecessarily be dependent upon the therapeutic effect to be achieved(treatment of infertility; contraception), and may vary with theparticular compound, the route of administration, and the age andcondition of the individual subject to whom the medicament is to beadministered.

In general, parenteral administration requires lower dosages than othermethods of administration which are more dependent upon adsorption.However, a dosage for humans preferably contains 0.0001-25 mg per kgbody weight. The desired dose may be presented as one dose or asmultiple subdoses administered at appropriate intervals throughout theday. In case of female recipients, doses may be administered atappropriate daily intervals throughout the menstrual cycle forfollicular support or as a single dose for ovulation induction. Thedosage as well as the regimen of administration may differ between afemale and a male recipient.

In case of in vitro or ex vivo applications, like in IVF applications,the compounds of the inventions are to be used in the incubation mediain a concentration of approximately 0.01-5 μg/ml.

The present invention thus also relates to pharmaceutical compositionscomprising a thieno[2,3-d]pyrimidine compound according to formula I inadmixture with pharmaceutically acceptable auxiliaries, and optionallyother therapeutic agents. The auxiliaries must be “acceptable” in thesense of being compatible with the other ingredients of the compositionand not deleterious to the recipients thereof.

Pharmaceutical compositions include those suitable for oral, rectalnasal, topical (including transdermal, buccal and sublingual), vaginalor parenteral (including subcutaneous, intramuscular, intravenous andintradermal) administration. The compositions may be prepared by anymethod well known in the art of pharmacy, for example, using methodssuch as those described in Gennaro et al., Remington's PharmaceuticalSciences (18th ed., Mack Publishing company, 1990, see especially Part8: Pharmaceutical Preparations and Their Manufacture).

Such methods include the step of bringing in association the activeingredient with any auxilliary agent. The auxilliary agent(s), alsonamed accessory ingredients, include those conventional in the art(Gennaro, supra), such as, fillers, binders, diluents, disintegrants,lubricants, colorants, flavoring agents and wetting agents.

Pharmaceutical compositions suitable for oral administration may bepresented as discrete dosage units such as pills, tablets or capsules,or as a powder or granules, or as a solution or suspension. The activeingredient may also be presented as a bolus or paste. The compositionscan further be processed into a suppository or enema for rectaladministration.

For parenteral administration, suitable compositions include aqueous andnon-aqueous sterile injection. The compositions may be presented inunit-dose or multi-dose containers, for example sealed vials andampoules, and may be stored in a freeze-dried (lyophilised) conditionrequiring only the addition of sterile liquid carrier, for example,water prior to use.

Compositions, or formulations, suitable for administration by nasalinhalation include fine dusts or mists which may be generated by meansof metered dose pressurized aerosols, nebulisers or insufflators.

The thieno[2,3-d]pyrimidine compounds of the invention can also beadministered in the form of implantable pharmaceutical devices,consisting of a core of active material, encased by a releaserate-regulating membrane. Such implants are to be applied subcutaneouslyor locally, and will release the active ingredient at an approximatelyconstant rate over relatively large periods of time, for instance fromweeks to years. Methods for the preparation of implantablepharmaceutical devices as such are known in the art, for example asdescribed in European Patent 0,303,306 (AKZO N.V.).

Thus, the compounds according to the present invention can be used forthe same clinical purposes as the native LH, with the advantage thatthey possess FSH activity, display altered stability properties and canbe administered differently.

The compounds of the present invention, represented by formula (I) cangenerally be prepared by nucleophilic substitution of halides (II)wherein Q=Cl or Br with (cyclic) secondary amines of formula (III) in anappropriate solvent such as N,N-dimethylformamide or THF at roomtemperature in the presence of a tertiary base such asN,N-diisopropylethylamine (DIPEA).

Derivatives of formula (II) wherein Q=Cl or Br can be prepared byregioselective acylation of meta aniline derivative (V) with acylchlorides of type (IV), wherein Q=Cl or Br in the presence of a tertiarybase such as N,N-diisopropylethylamine in a suitable solvent such asdichloromethane or THF.

Compound (V) is accessible by art-known reduction of the nitro functionin derivative (VI) using an appropriate reducing agent such as tin(II)chloride in a protic solvent such as ethanol in the presence ofhydrochloric acid at elevated temperature (J. Heilbron, J: Chem. Soc,1279 (1940)).

Thienopyrimidine (VI) can be prepared by condensation of carboxylic acid(VII) with tert-butyl amine under the influence of a coupling agent suchas O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate(TBTU) or bromotripyrrolidinophosphonium hexafluorophosphate (PyBrOP)and a tertiary base, e.g. N,N-diisopropylethylamine.

Saponification of ethyl ester (VIII) to the corresponding carboxylicacid (VII) takes place in the presence of a base such as lithiumhydroxide, potassium hydroxide or sodium hydroxide in aqueous dioxane atelevated temperature (80° C. to reflux).

Bicycle (VIII) is formed by substitution of chloride (X) with ethylmercaptoacetate under the agency of N,N-diisopropylethylamine, followedby base-catalyzed ring-closure of the intermediate thioether (IX). Thistype of thieno[2,3-d]pyrimidine ring formations has been described in:S. A. Abdel-Hady, M. A. Badawy, Y. A. Ibrahim, Sulfur Lett. 9, 101(1989) and S. Tumkevicius, Liebigs Ann., 1703 (1995).

Suitable conditions for the cyclization reaction are sodium ethoxide inethanol or N,N-diisopropylethylamine in toluene/ethanol (1/1, v/v) atreflux temperature.

The requisite chloroimine (X) can be synthesized following literatureprocedures as described for example by A. A. Santilli, D. H. Kim and S.V. Wanser, J. Heterocycl. Chem. 8, 445, 1971. According to thisprocedure, lactam (XI) is treated with POCl₃ at elevated temperature(80° C. to reflux) to give chloride (X). The addition of an appropriatesolvent, e.g. dioxane, and/or the addition of either PCl₅ orNN-dimethylaniline to the reaction mixture may result in shorterreaction times and higher yields of chloride (X).

An appropriate route towards lactam (XI) comprises the multicomponentcondensation of ethyl cyanoacetate with 3-nitro-benzaldehyde andS-methyl isothiourea in ethanol under the agency of a base such aspotassium carbonate at elevated temperature (60° C.).

Related procedures have been disclosed in: S. Kambe, K. Saito and H.Kishi, Synthesis, 287 (1979); A. M. Abd-Elfattah, S. M. Hussain and A.M. El-Reedy, Tetrahedron 39, 3197 (1983); S. M. Hussain, A. A.El-Barbary and S. A. Mansour, J. Heterocycl. Chem. 22, 169 (1985).

Methods to determine receptor binding as well as in vitro and in vivoassays to determine biological activity of gonadotropins are well known.In general, expressed receptor is contacted with the compound to betested and binding or stimulation or inhibition of a functional responseis measured.

To measure a functional response isolated DNA encoding the LH or the FSHreceptor gene, preferably the human receptor, is expressed in suitablehost cells. Such a cell might be the Chinese Hamster Ovary cell, butother cells are also suitable.

Preferably the cells are of mammalian origin (Jia et al, Mol.Endocrin.,5, 759-776, 1991).

Methods to construct recombinant LH or FSH expressing cell lines arewell known in the art (Sambrook et al., Molecular Cloning: a LaboratoryManual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, latestedition). Expression of receptor is attained by expression of the DNAencoding the desired protein. Techniques for site directed mutagenesis,ligation of additional sequences, PCR, and construction of suitableexpression systems are all, by now, well known in the art. Portions orall of the DNA encoding the desired protein can be constructedsynthetically using standard solid phase techniques, preferably toinclude restriction sites for ease of ligation. Suitable controlelements for transcription and translation of the included codingsequence can be provided to the DNA coding sequences. As is well known,expression systems are now available which are compatible with a widevariety of hosts, including prokaryotic hosts such as bacteria andeukaryotic hosts such as yeast, plant cells, insect cells, mammaliancells, avian cells and the like.

Cells expressing the receptor are then contacted with the test compoundto observe binding, or stimulation or inhibition of a functionalresponse.

Alternatively isolated cell membranes containing the expressed receptormay be used to measure binding of compound.

For measurement of binding radioactively or fluorescently labeledcompounds may be used. As reference compound human recombinant LH or FSHcan be used. In the alternative also competition binding assays can beperformed.

Another assay involves screening for LH or FSH receptor agonistcompounds by determining stimulation of receptor mediated cAMPaccumulation. Thus, such a method involves expression of the receptor onthe cell surface of a host cell and exposing the cell to the testcompound. The amount of cAMP is than measured. The level of cAMP will bereduced or increased, depending on the inhibitory or stimulating effectof the test compound upon binding to the receptor.

In addition to direct measurement of e.g. cAMP levels in the exposedcell, cells can be used which in addition to transfection with receptorencoding DNA are also transfected with a second DNA encoding a reportergene the expression of which responds to the level of cAMP. Suchreporter genes might be cAMP inducible or might be constructed in such away that they are connected to novel cAMP responsive elements. Ingeneral, reporter gene expression might be controlled by any responseelement reacting to changing levels of cAMP. Suitable reporter genes aree.g. LacZ, alkaline phosphatase, firefly luciferase and greenfluorescence protein. The principles of such transactivation assays arewell known in the art and are described e.g. in Stratowa, Ch, Himmler, Aand Czernilofsky, A. P., Curr.Opin.Biotechnol.6, 574 (1995).

For selecting active compounds on the LH or FSH receptor, testing at10⁻⁵ M must result in an activity of more than 20% of the maximalactivity when LH or FSH is used as a reference. Another criterion mightbe the EC₅₀ value, which must be <10⁻⁵ M, preferably <10⁻⁷ M.

The skilled artisan will recognize that desirable EC₅₀ values aredependent on the compound tested. For example, a compound with an EC₅₀,which is less than 10-5 M is generally, considered a candidate for drugselection. Preferably this value is lower than 10⁻⁷ M. However, acompound which has a higher EC₅₀, but is selective for the particularreceptor, may be even a better candidate.

Screening for LH receptor agonistic compounds can also be performed byusing a mouse Leydig cell bioassay (Van Damme, M., Robersen, D. andDiczfalusy, E., Acta Endocrinol. 77: 655-671 (1974), Mannaerts, B.,Kloosterboer, H. and Schuurs, A., Neuroendocrinology of reproduction. R.Rolland et al. Eds., Elsevier Science Publishers B.V., 49-58 (1987)). Inthis assay, stimulation of LH receptor mediated testosterone productioncan be measured in Leydig cells isolated from male mice.

FSH agonistic activity of compounds can also be determined in an ex vivomodel using cultured mouse follicles according to Nayudu, P. and Osborn,S. (J. Reproduction and Fertility 95, 349-362 (1992)). Therefore, mouseovarian follicles are isolated and cultured in the presence of FSHagonistic compounds to induce follicular growth. Measurements offollicular diameter and estradiol in the culture medium are indicativefor follicular growth.

To measure LH in vivo activity of compounds, ovulation induction inimmature mice can be studied. In this assay immature female mice areprimed with urinary FSH and approximately 48 hours later treated with aLH agonistic compound. The animals are killed after LH agonist treatmentand the number of ova in the oviduct is microscopically assessed.

To measure FSH in vivo activity of compounds immature female rats aretreated at 0, 8, 24 and 32 hours with a FSH agonistic compound to inducefollicular growth. At 52 hours after the start of the experiment theanimals are injected with hCG to induce ovulation. The animals arekilled 72 hours after the start of the experiment and the number of ovain the oviduct is microscopically assessed. In addition ovarian weightis determined.

The compounds of the present invention can be applied clinically inthose regimens where now LH or hCG is used. These include LHsubstitution among subjects with hypogonadal hypogonadism either male orfemale, midcycle administration to induce ovulation (ovulation induction(OI) or controlled hyperstimulation (COH) or stimulation of the corpusluteum.

The following examples are illustrative for the invention and should inno way be interpreted as limiting the scope of the invention.

EXAMPLES Example 1 tert-Butyl5-amino-2-methylthio-4-(3-(2-(azetidin-1-yl)-acetamido)-phenyl)-thieno[2,3-d]pyrimidine-6-carboxamide(a). 5-Cyano-4-(3-nitrophenyl)-2-methylthio-6-oxopyrimidine

A mixture of S-methylisothiourea sulfate (69.0 g), 3-nitrobenzaldehyde(75.0 g), ethyl cyanoacetate (56.0 ml) and potassium carbonate (72.5 g)in abs. EtOH (1500 ml) was stirred at 60° C. for 16 h. The reactionmixture was cooled to 0° C. in an ice bath. The resulting precipitatewas filtered off, washed with abs. EtOH and dissolved in hot water (100°C.). The solution was cooled to room temperature, acidified with 2N HClto pH 2 and cooled to 0° C. in an ice bath. The resulting precipitatewas filtered off and washed with ice water. Residual water in theprecipitate was removed by coevaporation with 1,4-dioxane.

Yield: 54.0 mg. MS-ESI: [M+H]⁺=289.0 TLC: R_(f)=0.3, silica gel,DCM/MeOH=9/1 (v/v).

(b). 6-Chloro-5-cyano-4-(3-nitrophenyl)-2-methylthio-pyrimidine

POCl₃ (100 ml) was added to a stirred solution of5-cyano-4-(3-nitrophenyl)-2-methylthio-6-oxopyrimidine (example 1(a),25.0 g) in dry 1,4-dioxane (300 ml). After 3 h at 90° C., the mixturewas cooled to room temperature and concentrated under reduced pressure.The residue was dissolved in 1,4-dioxane (100 ml) and the resultingsolution was cooled to 0° C. Ice water was cautiously added. Theresulting precipitate was filtered off and washed with water. Residualwater in the precipitate was removed by coevaporation with 1,4-dioxane.

Yield: 26.0 g. MS-ESI: [M+H]⁺=307.0 TLC: R_(f)=0.5, silica gel,heptane/EtOAc=3/2 (v/v).

(c). Ethyl5-cyano-4-(3-nitrophenyl)-2-methylthio-6-(ethoxycarbonylmethylthio)-pyrimidine

DIPEA (15.7 ml) was added to a stirred solution of ethyl2-mercaptoacetate (9.3 ml) and6-chloro-5-cyano-4-(3-nitrophenyl)-2-methylthio-pyrimidine (example1(b), 26.0 g) in a mixture of EtOH (250 ml) and DCM (250 ml). After 1 hat room temperature, 0.1N aq. HCl (500 ml) was added to the mixturewhich was then extracted with DCM (3*500 ml), dried (MgSO₄) andconcentrated under reduced pressure.

Yield: 28.0 g MS-ESI: [M+H]⁺=390.4 TLC: R_(f)=0.5, silica gel,heptane/EtOAc=3/2 (v/v).

(d). Ethyl5-amino-4-(3-nitrophenyl)-2-methylthio-thieno[2,3-d]pyrimidine-6-carboxylate

A mixture of ethyl5-cyano-4-(3-nitrophenyl)-2-methylthio-6-(ethoxycarbonylmethylthio)-pyrimidine(example 1(c), 28.0 g) and DIPEA (30 ml) in a mixture of toluene (150ml) and EtOH (150 ml) was stirred at reflux temperature (100° C.) for 16h. The mixture was then cooled to room temperature and concentratedunder reduced pressure. Residual DIPEA was removed by coevaporation withtoluene.

Yield: 28.0 g MS-ESI: [M+H]⁺=391.2 TLC: R_(f)=0.6, silica gel,heptane/EtOAc=3/2 (v/v).

(e). Ethyl5-amino-4-(3-aminophenyl)-2-methylthio-thieno[2,3-d]pyrimidine-6-carboxylate

EtOH (400 ml) was added to a mixture of ethyl5-amino-4-(3-nitrophenyl)-2-methylthio-thieno[2,3-d]pyrimidine-6-carboxylate(example 1(d), 28.0 g), concentrated aq. HCl (15 ml) and tin (II)chloride (41.0 g) in 1,4-dioxane (400 ml). The mixture was stirred at90° C. for 16 h. The mixture was then cooled to room temperature andconcentrated under reduced pressure. The residue was suspended, in EtOAc(1000 ml). 4N aq. NaOH was added to obtain a pH of 10-11. The mixturewas vigourously stirred and the organic layer was separated, dried(MgSO₄) and concentrated under reduced pressure.

Yield: 21.0 g MS-ESI: [M+H]⁺=361.0 TLC: R_(f)=0.6, silica gel,heptane/EtOAc=3/2 (v/v).

(f).5-Amino-4-(3-aminophenyl)-2-methylthio-thieno[2,3-d]pyrimidine-6-carboxylicacid

Potassium hydroxide (32.4 g) was added to a solution of ethyl5-amino-4-(3-aminophenyl)-2-methylthio-thieno[2,3-d]pyrimidine-6-carboxylate(example 1(e), 21.0 g) in a mixture of 1,4-dioxane (300 ml) and water(100 ml). After 16 h at 90° C., the mixture was cooled to 10° C. and 2Naq. citric acid (300 ml) was added under vigourous stirring. Theresulting precipitate was filtered off, washed with water (180 ml) anddried in vacuo.

Yield: 14.0 g MS-ESI: [M+H]⁺=333.0 TLC: R_(f)=0.5, silica gel,DCM/MeOH=9/1 (v/v).

(g). tert-Butyl5-amino-4-(3-aminophenyl)-2-methylthio-thieno[2,3-d]pyrimidine-6-carboxamide

TBTU (16.1 g) was added to a solution of5-amino-4-(3-aminophenyl)-2-methylthio-thieno[2,3-d]pyrimidine-6-carboxylicacid (example 1(f), 14.0 g), DIPEA (17.4 ml) and tert-butylamine (7.3 g)in DCM/DMF (1/1, v/v, 250 ml). After 3 h at room temperature, themixture was washed with sat. aq. NaHCO₃ (3*100 ml), 0.1 N aq. HCl (100ml) and water (100 ml). The organic layer was concentrated under reducedpressure. The crude product was purified by crystallization from warmabs. EtOH (300 ml).

Yield: 10.5 g MS-ESI: [M+H]⁺=388.2 HPLC: R_(t)=30.72 min, Luna C-18(2),5 μm, 250*2.0 mm, detection UV=210 nm, oven temperature=40° C.,flow=0.25 ml/min, eluent water/ACN/MeOH=90/9.5/0.5 to 0/95/5, runtime=50 min.

(h). tert-Butyl5-amino-2-methylthio-4-(3-(2-bromoacetamido)-phenyl)-thieno[2,3-d]pyrimidine-6-carboxamide

Bromoacetylchloride (615 mg) was added to a solution of tert-butyl5-amino-2-methylthio-4-(3-aminophenyl)-thieno[2,3-d]-pyrimidine-6-carboxamide(example 1(g), 1.08 g) and DIPEA (2.43 ml) in dry DCM (20 ml). After 3 hat room temperature, the mixture was diluted with DCM, washed with sat.aq. NaHCO₃, dried (MgSO₄) and concentrated under reduced pressure. Thecrude product was purified by chromatography on silica gel usingheptane/EtOAc=3/2 (v/v) as eluent.

Yield: 910 mg MS-ESI: [M+H]⁺=510.2 TLC: R_(f)=0.3, silica gel,heptane/EtOAc=3/2 (v/v).

(i). tert-Butyl5-amino-2-methylthio-4-(3-(2-(azetidin-1-yl)-acetamido)-phenyl)-thieno[2,3-d]pyrimidine-6-carboxamide

tert-Butyl5-amino-4-(3-(2-bromoacetamido)-phenyl)-2-methylthio-thieno[2,3-d]pyrimidine-6-carboxamide(example 1(h), 91 mg) was added to a solution of azetidine hydrochloride(120 mg) and N,N-diisopropylethylamine (0.25 ml) in DCM (5 ml). After 16h at room temperature, the mixture was washed with sat. aq. NaHCO₃,dried (MgSO₄) and concentrated under reduced pressure. The crude productwas purified by HPLC using a Luna C-18 column with the followinggradient: 0.1% aq.TFA+10% aq. ACN/ACN=90/10 to 10/90 in 30 min. Thetitle compound was then lyophilized from water with 0.1% TFA.

Yield: 56 mg (TFA-salt) MS-ESI: [M+H]⁺=485.2 HPLC: R_(t)=13.45 min,column Luna C-18(2), 3 μm, 100*2.0 mm, detection UV=210 nm, oventemperature=40° C., flow=0.25 ml/min, eluent phosphate buffer 50 mM pH2.1/water/ACN=10/70/20 to 10/10/80 (v/v), run time=20 min.

Example 2 tert-Butyl5-amino-2-methylthio-4-(3-(2-(morpholin-4-yl)-acetamido)-phenyl)-thieno[2,3-d]pyrimidine-6-carboxamide

Morpholine (5:0 ml) was added to a solution of tert-butyl5-amino-4-(3-(2-bromoacetamido)-phenyl)-2-methylthio-thieno[2,3-d]pyrimidine-6-carboxamide(example 1(h), 1.0 g) in THF (50 ml). After 16 h at room temperature,the mixture was concentrated under reduced pressure. The residue waspurified by column chromatography on silica gel using DCM/MeOH=9/1 aseluent. The crude product was further purified by HPLC using a Luna C-18column with the following gradient: 0.1% aq. TFA/water/ACN=3/97/0 to3/7/90 in 30 min. The pure title compound was lyophilized from a mixtureof 0.1% aq. TFA and water.

Yield: 215 mg (TFA-salt) MS-ESI: [M+H]⁺=515.2 HPLC: R_(t)=20.62 min,Luna C-18 (2), 5 μm, 150*2 mm, detection UV=210 nm, oven temperature=40°C., flow=0.25 ml/min, eluent phosphate buffer 50 mM pH2.1/water/ACN/MeOH=10/72/17/1 to 10/18/68/4 (v/v), run time=40 min.

Example 3 tert-Butyl5-amino-2-methylthio-4-(3-(2-(thiomorpholin-4-yl)-acetamido)-phenyl)-thieno[2,3-d]pyrimidine-6-carboxamide

Thiomorpholine (2.16 ml) was added to a solution of tert-butyl5-amino-2-methylthio-4-(3-(2-bromoacetamido)-phenyl)-thieno[2,3-d]pyrimidine-6-carboxamide(example 1(h), 1.09 g) in DCM (50 ml). After 16 h at room temperature,the mixture was diluted with DCM, washed with sat. aq. NaHCO₃, dried(MgSO₄) and concentrated under reduced pressure. The crude product waspurified by HPLC using a Luna C-18 column with the following gradient:0.1% aq. TFA+10% aq. ACN/ACN=100/0 to 10/90 in 30 min. The pure titlecompound was lyophilized from water acidified with aq. 1N HCl.

Yield: 816 mg MS-ESI: [M+H]⁺=531.2 HPLC:R_(t)=14.72 min, column LunaC-18(2), 3 μm, 100*2 mm, detection UV=210 nm+254 nm, oventemperature=40° C., flow=0.25 ml/min, eluent phosphate buffer 50 mM pH2.1/water/ACN/MeOH=10/72/17/1 to 10/18/68/4(v/v), run time=20 min.

Example 4 tert-Butyl5-amino-2-methylthio-4-(3-(2-(piperidin-1-yl)-acetamido)-phenyl)-thieno[2,3-d]pyrimidine-6-carboxamide

Piperidine (3.0 ml) was added to a solution of tert-butyl5-amino-4-(3-(2-bromoacetamido)-phenyl)-2-methylthio-thieno[2,3-d]pyrimidine-6-carboxamide(example 1(h), 1.0 g) in CH₂Cl₂ (50 ml). After 16 h at room temperature,the mixture was concentrated under reduced pressure. The residue waspurified by column chromatography on silica gel using DCM/MeOH=9/1 aseluent. The crude product was further purified by HPLC using a Luna C-18column with the following gradient: 0.1% aq. TFA/ACN=100/0 to 10/90 in30 min. The pure title compound was lyophilized from a mixture of 0.1%aq. TFA and water.

Yield: 851 mg (TFA-salt) MS-ESI: [M+H]⁺=513.2 HPLC: Rt=37.3 min, LunaC-18 (2), 5 μm, 150*2 mm, detection UV=210 nm, oven temperature=40° C.,flow=0.25 ml/min, eluent phosphate buffer 50 mM pH2.1/water/ACN=20/60/20 to 20/0/80 (v/v), run time=40 min.

Example 5 tert-Butyl5-amino-2-methylthio-4-(3-(2-(pyrrolidin-1-yl)-acetamido)-phenyl)-thieno[2,3-d]pyrimidine-6-carboxamide

Pyrrolidine (3.0 ml) was added to a solution of tert-butyl5-amino-4-(3-(2-bromoacetamido)-phenyl)-2-methylthio-thieno[2,3-d]pyrimidine-6-carboxamide(example 1(h), 1.0 g) in CH₂Cl₂ (50 ml). After 16 h at room temperature,the mixture was concentrated under reduced pressure. The residue waspurified by column chromatography on silica gel using DCM/MeOH=9/1 aseluent. The crude product was further purified by HPLC using a Luna C-18column with the following gradient: 0.1% aq. TFA/ACN=100/0 to 10/90 in30 min. The pure title compound was lyophilized from a mixture of 0.1%aq. TFA and water.

Yield: 616 mg (TFA-salt) MS-ESI: [M+H]⁺=499.2 HPLC: Rt=37.5 min, LunaC-18 (2), 5 μm, 150*2 mm, detection UV=210 nm, oven temperature=40° C.,flow=0.25 ml/min, eluent phosphate buffer 50 mM pH2.1/water/ACN=20/60/20 to 20/0/80 (v/v), run time=40 min.

Example 6 tert-Butyl5-amino-2-methylthio-4-(3-(2-(piperidin-1-yl)-acetamido)-phenyl)-thieno[2,3-d]pyrimidine-6-carboxamide

Piperazine (2.5 g) was added to a solution of tert-butyl5-amino-4-(3-(2-bromoacetamido)-phenyl)-2-methylthio-thieno[2,3-d]pyrimidine-6-carboxamide(example 1(h), 1.0 g) in CH₂Cl₂ (50 ml). After 16 h at room temperature,the mixture was concentrated under reduced pressure. The residue waspurified by column chromatography on silica gel using DCM/MeOH=7/1 aseluent. The crude product was further purified by HPLC using a Luna C-18column with the following gradient: 0.1% aq. TFA/ACN=100/0 to 10/90 in30 min. The pure title compound was lyophilized from a mixture of 0.1%aq. TFA and water.

Yield: 766 mg (bis TFA-salt) MS-ESI: [M+H]⁺=514.4 HPLC: Rt=33.7 min,Luna C-18 (2), 5 μm, 150*2 mm, detection UV=210 nm, oven temperature=40°C., flow=0.25 ml/min, eluent phosphate buffer 50 mM pH2.1/water/ACN=20/60/20 to 20/0/80 (v/v), run time=40 min.

Example 7 CHO-LH and CHO—FSH in vitro Bioactivity

LH agonistic activity of compounds were tested in Chinese Hamster Ovary(CHO) cells stably transfected with the human receptor and cotransfectedwith a cAMP responsive element (CRE)/promotor directing the expressionof a firefly luciferase reporter gene. Binding of ligand to theGs-coupled LH receptor will result in an increase of cAMP, which in turnwill induce an increased transactivation of the luciferase reporterconstruct. The luciferase signal was quantified using a luminescencecounter. For test compounds, EC₅₀ values (concentration of test compoundcausing half-maximal (50%) stimulation) were calculated. For thatpurpose the software program GraphPad PRISM, version 3.0 (GraphPadsoftware Inc., San Diego) was used.

In a similar way FSH agonistic activity of compounds was tested in CHOcells transfected with the luciferase reporter gene and the human FSHreceptor. Results are shown in Table 1.

In vivo Bioactivity

To measure in vivo activity of LH/FSH receptor agonistic compoundsovulation induction in immature mice was studied. In this assay immaturefemale mice were primed with urinary FSH (Humegon 12.5 IU/animal).Approximately 48 hours later the animals were treated with a LH/FSHagonistic compound at a dose-level of 50 mg/kg. The animals were killed24 hours after LH/FSH agonist treatment and the number of ova in theoviduct was microscopically assessed. Results are shown in Table 1.

TABLE 1 Example EC50 CHO EC50 CHO no. animals mean no. of ova no. NameLHR (M) FSHR (M) tested (50 mg/kg p.o.) 1 tert-Butyl5-amino-2-methylthio-4-(3-(2- 3.86E−09 4.62E−07 15 1.6(azetidin-1-yl)-acetamido)-phenyl)-thieno[2,3-d]pyrimidine-6-carboxamide 2 tert-Butyl5-amino-2-methylthio-4-(3-(2- 2.24E−09 4.20E−08 10 9.3(morpholin-4-yl)-acetamido)-phenyl)-thieno[2,3-d]pyrimidine-6-carboxamide 3 tert-Butyl5-amino-2-methylthio-4-(3-(2- 8.03E−09 1.04E−06 10 19.8(thiomorpholin-4-yl)-acetamido)-phenyl)-thieno[2,3-d]pyrimidine-6-carboxamide 4 tert-Butyl5-amino-2-methylthio-4-(3-(2- 6.63E−09 2.01E−07(piperidin-1-yl)-acetamido)-phenyl)-thieno[2,3-d]pyrimidine-6-carboxamide 5 tert-Butyl5-amino-2-methylthio-4-(3-(2- 6.68E−09 4.80E−07(pyrrolidin-1-yl)-acetamido)-phenyl)-thieno[2,3-d]pyrimidine-6-carboxamide 6 tert-Butyl5-amino-2-methylthio-4-(3-(2- 3.17E−09 1.50E−07(piperazin-1-yl)-acetamido)-phenyl)-thieno[2,3-d]pyrimidine-6-carboxamide

1. A thieno[2,3-d]pyrimidine according to

formula I, or a pharmaceutically acceptable salt thereof, wherein R1 andR2 together with the nitrogen atom to which they are bonded form a ringhaving 2-6 carbon atoms, optionally containing one or more heteroatomsselected from N, O and/or S.
 2. A compound selected from the groupconsisting of tert-butyl5-amino-2-methylthio-4-(3-(2-(azetidin-1-yl)-acetamido)-phenyl)-thieno[2,3-d]pyrimidine -6-carboxamide; tert-butyl5-amino-2-methylthio-4-(3-(2-(morpholin-4-yl)-acetamido)-phenyl)-thieno[2,3-d]pyrimidine-6-carboxamide; tert-butyl5-amino-2-methylthio-4-(3-(2-(thiomorpholin-4-yl)-acetamido)-phenyl)-thieno[2,3-d]pyrimidine-6-carboxamide;tert-butyl5-amino-2-methylthio-4-(3-(2-(piperidin-1-yl)-acetamido)-phenyl)-thieno[2,3-d]pyrimidine-6-carboxamide; tert-butyl5-amino-2-methylthio-4-(3-(2-(pyrrolidin-1-yl)-acetamido)-phenyl)-thieno[2,3-d]pyrimidine-6-carboxamideand tert-butyl 5-amino-2-methylthio-4-(3-(2-(piperazin-1-yl)-acetamido)-phenyl)-thieno[2,3-d]pyrimidine-6-carboxamideor a pharmaceutically acceptable salt thereof.
 3. A pharmaceuticalcomposition, comprising: the thieno[2,3-d]pyrimidine compound accordingto claim 1 or a pharmaceutically acceptable salt thereof, and apharmaceutically acceptable auxiliary.
 4. A method to treat fertilitydisorders in patients in need of luteinizing hormone and folliclestimulating hormone treatment, comprising: administering an effectiveamount of the compound or pharmaceutically acceptable salt thereofaccording to claim
 1. 5. A pharmaceutical composition, comprising: thethieno[2,3-d]pyrimidine compound according to claim 2 or apharmaceutically acceptable salt thereof, and a pharmaceuticallyacceptable auxiliary.
 6. A method to treat fertility disorders inpatients in need of luteinizing hormone and follicle stimulating hormonetreatment, comprising: administering an effective amount of the compoundor a pharmaceutically acceptable salt thereof according to claim
 2. 7.The method of claim 4, wherein the fertility disorder is selected fromthe group consisting of hypogonadism and male infertility.
 8. The methodof claim 4, wherein the fertility disorder is hypogonadism.
 9. Themethod of claim 4, wherein the fertility disorder is male infertility.10. The method of claim 6, wherein the fertility disorder is selectedfrom the group consisting of hypogonadism and male infertility.
 11. Themethod of claim 6, wherein the fertility disorder is hypogonadism. 12.The method of claim 6, wherein the fertility disorder is maleinfertility.
 13. A method for treating infertility by ovulationinduction or by controlled hyperstimulation, the method comprisingadministering an effective amount of the compound or pharmaceuticallyacceptable salt thereof according to claim
 1. 14. A method for treatinginfertility by ovulation induction or by controlled hyperstimulation,the method comprising administering an effective amount of the compoundor pharmaceutically acceptable salt thereof according to claim 2.